Mitochondrial signaling maintains and integrates essential aerobic functions in energy, fatty acid, amino acid, heme and steroid hormone metabolism with other cell functions. Poisons and environmental toxicants often target specific mitochondrial enzymes and transport systems, resulting in stress signaling, activation of cell death (necrosis or apoptosis), mitochondrial failure and removal (mitophagy), and increased mitochondrial biogenesis. During these processes, ROS contributes to pathology and toxicity by impacting transcription or activating cell death. At lower rates, ROS selectively oxidizes cysteine (Cys) residues in target proteins, thereby integrating with the broader system of mitochondrial metabolic signaling, which modulates many cellular functions. With this complexity, challenges exist to discriminate mitochondrial signaling from other sites of origin. This project will develop and test an integrated redox proteome x metabolome x transcriptome (RMT) tool for study of mitochondria-cell signaling networks in environmental toxicology. The project will use manganese (Mn), an environmental metal ion (Mn2+) known to have beneficial as well as toxic effects in mitochondria via ROS, for tool development. The research strategy will involve controlled dose response from low to high Mn in neuronal cells. Mitochondrial reactive oxygen species (ROS) generation and mitochondrial redox proteomics will be used to calibrate mitochondrial oxidation. A combined transcriptome-, metabolome-wide association study will be used to elucidate signaling networks. In the R21 phase (Aim 1), computational methods and bioinformatics will identify central hubs connecting the mitochondrial redox proteomics changes with the metabolome-transcriptome association network. In the R33 phase (Aim 2), the algorithms for this mitochondrial RMT will provide the basis to create software and online tools to facilitate identification of mitochondrial-cellular signaling networks by individuals with limitd computational skills. The team will collaborate with HERCULES (Health and Exposome Research Center: Understanding Lifetime Exposures, supported by NIEHS) investigators and their trainees to test the usability of RMT by individuals without specific training in computationl biology. Further development and testing of the RMT tools will be done in Aim 3, using a cellular model for Huntington's disease (HD) and non-neuronal cells to test the ability to identify variations in mitochondria-cell signaling networks, mouse models with altered sensitivity to mitochondrial toxicity to test translation to in vivo research, and alternate environmental stressors to test general utility of the RMT tools. The development utilizes contemporary technologies and expertise of the investigative team and will fulfill critical needs to measure and understand mitochondria-cellular signaling in response to environmental exposures in the context of complex disease mechanisms. The results obtained from RMT will be global in nature yet inherently mechanistic. The design will enable general use to address challenges associated with the study of mitochondria as targets of environmental disease.